Discussion On The Method Inducing Retinal Stem Cell Differentiate Into Ganglion/Progenitor Cell And Study Of The Effect Of SDF-1on The Regulation Of RGPC Orient Immigration

Objective：Injury of optic nerve is a common type in clinic which cause ablepsia usually. Theoptic nerve which belongs to central nervous is composed of retinal ganglioncells(RGCs)and its’ axon and difficult to reborn after injury. So far there are no effectivetreatments for these injury disease, comprehensive treatments of hormones impaction、surgical treatment and adjuvant therapy with neurotrophic factors are mainly used. Theinjury of optic nerve causes visual dysfunction because of the loss and apoptosis of RGCs,thus, reducing the apoptosis and increasing survival rate of RGCs are key points for therapy.As one way of cell replacement, the development of stem cell transplantation hasbrought us hope in optic nerve repair and regeneration after injury. Stem cell is able to copyitself by dividing and differentiate into different cells in different microenvironment andtriggering conditions. Retinal stem cells(RSCs) belong to adult stem cells and candifferentiate into RGCs, it’s been studied as seed cells in transplantation for the repairtreatment after optic nerve injury. However, there are many arguments on wheather inducestem cell into target cell in vitro first then transplant or transplant first then differentiate intotarget cell under the induction of tissue microenvironment at present.We are hoping to find a cell population which has not only multiplication capacity asstem cells but also partial characteristic of RGCs and expresses mature RGCs’ specificmarkers negatively, named retinal ganglion progenitor cell(RGPC).It can avoid theuncertainty of differentiation after transplant and have particular multiplication capacity andfigurability, which would provide a new help to cure optic nerve injury by celltransplantation.Cell therapy works only after transplanted cells migrate to damage area and combine to receptor tissue. Stromal cell-derived factor1(SDF-1) have migration effects on many typesof cells, studies show that it has important effects on stem cells’ migration.We are looking for the method of inducing stem cells differentiate into retinal ganglionprogenitor cells in vitro and discussing whether SDF-1can increase transplantation rate bypromoting directed migration to provide a new way of cell transplantation replacementtherapy to the optic nerve reborn after injury.Methods：Part I: Discussion on the method of inducing retinal stem cells differentiate intoRGCs and study of filtrating retinal ganglion progenitor cells1.Use E13.5LE embryo rat, isolate retinal stem cells and regenerate. Identify stem cellmark after2generation.2. Use flow cytometry to identify the positive rate of thy1.1after inducing14d’sdifferentiation of P2RSCs respectively in DAPT Group、BDNF group、DAPT unit BDNFgroup and5%FBS control group.3.Identify the stem cell markers nestin、ki67and RGCs markers math5、brn3d andthy1.1after using DAPT unit BDNF differential medium to induce P2RSCs1d、3d and5d.Part II: Research on the effect of SDF-1regulating RGPC migrated oriented1.DAPT unit BDNF differential medium induces P2RSCs differentiating3d andidentify RGPC marks and the expression of CXCR4receptor on the surface membrane ofcells.2.Set SDF-1group、SDF-1unit CXCR4blocker group and control group, observe themigration of each.3.Set experimental group of different SDF-1concentration and blank control group,observe the migration of each.Results：Part I: Discussion on the method of inducing retinal stem cells differentiate intoRGCs and study of filtrating retinal ganglion progenitor cells1.P2RSCs related markers chx10、nestin and ki67express positive and early agedRGCs markers math5express positive, ripe RGCs marker thy1.1express negative2.The positive rate of each experimental group compare with control group arestatistically significant, DAPT unit BDNF group has the highest thy1.1positive rate and is statistically significant with each group.3.After using DAPT uniting BDNF to induce P2RSC differentiated for3days,parts of cells express nestin、ki67、math5and brn3b positively while express thy1.1negatively. These cells are RGPC which have proliferation ability as stem cells and earlyRGC markers while have not yet became mature RGC.Part II: Research on the effect of SDF-1regulating RGPC migrated oriented1. After using DAPT uniting BDNF to induce P2RSC differentiated for3days,CXCR4receptor on the surface membranes of RGPC express Positive.2. SDF-1can induce RGPC to migrate oriented, while AMD3100can reduce the effectthrough blocking the combination of SDF-1and CXCR4.3. The effect that SDF-1induce RGPC to migrate oriented is regulated bythe concentration of SDF-1within certain limits, it effects more significant when theconcentration increase. It displayed the maximum efficacy at500ng/ml SDF-1concentration while the migration index is the highest.Conclusion：This research studied the method to increase the differentiation from induced RSC intoRGC in vitro, and screened the rough time when RGPC appear. In addition it also studiedthe effect of SDF-1on the regulation of RGPC orient immigration.Using DAPT uniting BDNF can increase the differentiation from induced RSC intoRGC, meanwhile RGPC appear after3days’ induction.SDF-1,combine with receptor CXCR4,can induce RGPC to migrate oriented, whichcould be blocked by AMD3100.The effect that SDF-1induce RGPC to migrate orientedenhanced with rise in concentration within certain limits, and it displayed the maximumefficacy at500ng/ml SDF-1concentration.